Method and Apparatus for the Processing and/or Analysis and/or Selection of Particles, in Particular, Biological Particles

ABSTRACT

Methods and apparatus are described for the processing (for example washing, incubation, etc.) of particles in which the particles suspended in a first fluid are introduced under laminar flow conditions into at least one first microchamber or first region of the same, in which a second fluid is introduced under laminar flow conditions into at least one second region of the microchamber or of a second microchamber, in such a way as not to mix with the first fluid, and in which at least one field of force acting on the particles is activated in the microchamber(s), to provoke a shift of the particles alone in a predetermined direction and to transfer the same in suspension into the second fluid; an apparatus is preferably used including at least three microchambers n microchambers arranged in sequence with each other in one direction and each connected with the microchamber immediately before it and after it with two orifices offset from each other in a direction perpendicular to the direction of sequence of the microchambers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.12/294,860 filed Sep. 26, 2008, which is the U.S. national phase ofPCT/IB2007/000751 filed Mar. 26, 2007, which claims the priority benefitof Italian Patent Application No. TO2006A000226 filed Mar. 27, 2006, theentire respective disclosures of which are incorporated herein byreference.

BACKGROUND

1. Technical Field

The present invention concerns methods and miniaturised apparatus forthe processing/manipulation of particles. The invention is appliedprincipally in the implementation of biological protocols on cellsamples in reduced volumes, as is often required by a miniaturisedanalysis approach.

2. State of the Art

In biology, the centrifuge is one of the instruments most used for theprocessing of cell samples, in particular with reference to certainstages of the process such as washing the cells. This is necessary forexample when starting from a culture, for which it is desirable toremove the culture medium and re-suspend the cells in a saline solution,or when, after an incubation phase with antibodies and/or otherreagents, one wants to wash away the excess antibodies and re-suspendthe cells in a saline buffer, etc. The potential advantage that can beachieved with the reduction of the volumes involved has led to theminiaturisation of these stages of the process, which has stimulated thedevelopment of various methods of processing/manipulation.

In Seger et al. Cell immersion and cell dipping in microfluidic devices,LabChip, 2004, 4, 148-151, pressure-controlled liquid flows are used toexchange cells from one suspension buffer to another (washing), or toimmerse cells originally present in a first buffer in a second bufferfor a controlled time (incubation or reagent-sampling). The disadvantageof this approach is that it is necessary to maintain a controlled flowof the various liquids involved. This implies a greater consumption ofreagents and a greater complexity of the system, linked with thenecessity of producing pressure to flush (move) the liquids.

A similar approach is used by EVOTEC to submit single cells trapped indielectrophoresis cages to particular reagents, but here too it isnecessary to have controlled flows of reagents.

The patent application PCT/WO 00/69565 a G. Medoro describes anapparatus and a method for the manipulation and identification ofparticles making use of closed cages with dielectrophoretic potential.The method described teaches how to control the position of eachparticle independently of all the others in a two-dimensional space. Theforce used to trap the particles in suspension is negativedielectrophoresis. The individual control of the manipulating operationsis achieved by the programming of memory elements and circuitsassociated with each element of an array of electrodes and sensorsintegrated in the same substratum.

The U.S. Pat. No. 6,294,063 Becker et al. describes a method andapparatus for the manipulation of packages of solid, liquid or gaseousbiological material by means of a distribution of programmable forces.

In the application for an Italian patent BO2005A000481, Medoro et al.,some methods are given for manipulating particles with arrays ofelectrodes, and some methods and apparatus for identifying them, whichare however similar to those of the already mentioned patent PCT/WO00/69565.

Although it does not allow a precise control of the position of theparticles, it is also possible to use Travelling Wave dielectrophoresisto shift the particles.

In other cases electrodes are not necessary to manipulate particlessituated inside the microchambers.

Then there are many known methods of generating optical traps (opticaltraps, optical tweezers), which are based on the differences of therefraction index of the particles with respect to the suspension medium(for example A. Ashkin et al, Optical trapping and manipulation ofsingle living cells using infrared laser beams, Nature, 330(6150) (1987)769.)

In this case the particles are typically trapped in optical intensitymaximums, generated for example by focussing a laser beam through thelens of a microscope. The manipulation of a multiplicity of particlesmay be obtained with various optical methods in the prior art, based onsimilar principles, for example using arrays of VCSEL (Vertical CavitySurface Emitting Laser), or using holographic optical traps.

Other techniques are known, which combine the projection of images withluminous intensity gradients, the use of an electric field in the liquidand a device with a photosensitive substratum, to create so-calledOpto-Electronic cages (for example U.S. Pat. No. 6,387,707B1 assigned toSeul et Al., or Pei Yu Chiou et al, “Massively parallel manipulation ofsingle cells and microparticles using optical images”, Nature Vol 436,21 Jul. 2005, pp 370-372). In practice a dielectrophoretic field isrealised, controlled non by the shape of the electrodes but by the imageprojected on the photosensitive substratum. In this way it is thereforepossible to manipulate the particles present in the liquid.

Lastly other techniques are known for moving particles. In S. Gaugiran,et al., “Optical manipulation of microparticles and cells on siliconnitride waveguides,” Opt. Express 13, 6956-6963 (2005), some particles(artificial or biological such as yeasts and cells) are pushed alongwave guides be the radiation pressure of the evanescent field of a laserwhich is spread inside the guide itself.

The international patent application in the name of the same Applicant,filed on 22 Mar. 2006 and concerning a method and an apparatus formaking the characterisation and/or the count of particles of any type,for example biological particles such as cells or their parts, in whichthe control of the position of each particle present in a sample is usedin order to shift those particles in a deterministic or statistical way,to detect their presence and/or characterise the type with opticalintegrated and/or impedenziometric sensors. In particular non uniformfields of force are used, variable times and optical or impedenziometricsensors located under or close to an array of electrodes, integratedwith them in a single chip. The fields of force may have positive ornegative dielectrophoresis, electrophoresis or electro-hydrodynamicmovements, characterised by a set of stable points of equilibrium.

However, among the methods reported in the above-mentioned inventionsconcerning micromanipulators of cells/particles based ondielectrophoresis or other optical or opto-electronic techniques,although a flow is not necessary, no method is contemplated for allowingthose typical operations such as washing and/or incubation of the cellsor particles, as is necessary in many biological protocols.

SUMMARY

The aim of the present invention is to supply a method and an apparatusfor carrying out multiple operations typically performed, up till now,on a macroscopic scale with test tubes and centrifuges, performing theminstead in a miniaturised way and/or with a lower consumption ofreagents and/or a greater delicacy of action on the cells/particlesand/or a greater efficiency of recovery of the processed cells and/or agreater control of the incubation/washing time and/or a greaterautomation and/or the extraction of particular information on individualcells.

Here and below, the terms “particles” or “particle” are used to indicatemicrometric or nanometric entities, natural or artificial, such ascells, subcellular components, viruses, liposomes, niosomes, microbeads(microspheres) and nanobeads, or even smaller entities such asmacro-molecules, proteins, DNA, RNA, etcetera, as well as drops of afluid that cannot be mixed in a suspension medium, for example oil inwater, or water in oil, or even drops of liquid in a gas (such as waterin air) or, again, bubbles of gas in a liquid (such as air in water).

The term cell will sometimes be used, but where not specified otherwiseit must be understood as a non limiting example of “particles”understood in the sense described more fully above.

According to the present invention, a method and an apparatus aretherefore supplied for realizing washing, incubation, and complexoperations (for example marking of cells with antibodies and/ormicrospheres) as defined in the claims.

In particular non uniform fields of force are used, variable times and,optionally, integrated sensors, preferable of the optical type.

The fields of force may, for example, have positive or negativedielectrophoresis, electrophoresis or electro-hydrodynamic movements,characterised by a set of points of stable equilibrium for the particles(solid, liquid or gaseous).

In this way the limitations of the prior art are overcome by the presentinvention.

The implementation of the method according to the invention does notrequire pumps or flows of liquid generated otherwise.

Unlike centrifuges, the cells are subjected to lower stresses linkedwith the pressure exerted by the other cells in the pellet, or withfriction with the walls of the test tube.

The method may be realised with miniaturised systems and the consumptionof reagents may be drastically reduced, also because it is not necessaryto support a flow of reagents.

Lastly, unlike the approaches that use flows to move the particles, byusing a controlled field of force to move the particles from one bufferto the other it is possible to control precisely, one particle at atime, the time each of these remains exposed to each reagent.

Lastly the apparatus realised according to the invention allows theimplementation of the method of the invention in a particularlyadvantageous way.

Further characteristics and advantages of the invention will be clearfrom the following description of some of its non limiting embodiments,with reference to the figures in the attached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an embodiment of a washing method realised according to theinvention compatible with the use of forces acting on the particles inonly one direction;

FIG. 2 shows an embodiment of a washing method according to theinvention which requires the manipulation of particles in twodimensions;

FIG. 3 shows an embodiment of a particle incubation method whichrequires the manipulation of particles in two dimensions;

FIG. 4 shows a first apparatus realised according to the presentinvention;

FIG. 5 shows a second apparatus realised according to the presentinvention.

DETAILED DESCRIPTION

The aim of the present invention is to supply a method and an apparatusfor carrying out multiple operations typically performed on amacroscopic scale with test tubes and centrifuges, but performing themin such a way as to have one or more of the following advantages:

-   -   miniaturisation;    -   lower consumption of reagents;    -   greater delicacy on the cells/particles;    -   greater efficiency of recovery of the processed cells;    -   greater control of the incubation/washing time;    -   greater automation;    -   integration of various steps of the process in a single        integrated device;    -   extraction of particular characteristic information on the        individual cells (e.g. dynamics of reagent uptake, variations of        volume, lysis) which are impossible with other methods.

The method of the invention is based on the use of a non uniform fieldof force (F) with which to attract single particles or groups ofparticles (BEADS) towards positions of stable equilibrium (CAGES). Thisfield may be, as an illustrative example without limitation, a field ofdielectrophoresis (DEP), negative (NDEP) or positive (PDEDP), or anelectrophoretic field (EF), an optophoretic field, etc.

The identification of the effects on the particle resulting from itsimmersion in a second buffer may concern one of the following aspects,or combinations of the same:

-   -   the identification of fluorescence    -   the identification of absorbance, transmittance or other optical        properties;    -   the identification of variations of volume or other physical        properties;    -   the identification of impedenziometric properties;    -   the identification of lysis of the particle.

Each of these observations may be carried out optionally at the level ofa single cells, and with time resolution such as to appreciate thevariation dynamics of the parameters studied.

Moreover, this information may be used not only to characterise theeffects of the buffer on the particles, but to make a selective recoveryof the particles with definite characteristics.

For this purpose the measurement of the impedance variation isprincipally used, and/or the measurement of the variation of lightintensity, transmitted, diffused or emitted in fluorescence.

Generation of Forces

There are various methods for generating forces to shift particles, asdescribed previously in the state of the art.

For the sake of simplicity, below is considered purely as an example,and therefore without limitation for the purposes of the presentinvention, the use of closed cages with negative dielectrophoresis asthe activating force for the description of the methods and apparatus(for which it is necessary to use a cover acting as an electrode) of theinvention. To experts of the sector with ordinary skills it is clearthat is possible to generalise the methods and apparatus described belowfor the use of different activating forces, and different types ofparticles.

The common characteristics of the forces which allow particles to bemanipulated according to the present invention is linked to the factthat these forces act mainly on the particle, moving the latter butnot—or to a very limited extent—the liquid in which it is immersed.

The use of one or the other force is relevantly indifferent, unlessfluorescent markers are involved. In these cases it is preferable to useforces based only on electric fields, so as not to cause photo-bleachingof the fluorophors, which could occur when applying the lightingotherwise necessary using optical or electro-optical methods.

Sensors Used

Also for the sake of simplicity, reference will be made below only tothe case of optical sensors, which allow the measurement of the opticalpower acting on a photodiode integrated with the electrodes. To expertsof the sector with ordinary skills it is clear that is possible togeneralise, in the various cases, the methods and apparatus describedbelow also for the alternative or combined use of integratedimpedenziometric sensors.

It is also clear to the expert of the sector with ordinary skills inwhich cases the use of integrated sensors is beneficial and in whichcases they are not necessary.

Method for Washing Particles

Reference is made to FIG. 1 and FIG. 2.

The method exploits the fact that when two liquids (L1, L2), even of atype that generally mix together, are introduced into at least onemicrochamber (for example CH1, FIG. 1), in such a way as to realise alaminar flow for each liquid during the phase in which it is introducedinto a predetermined region of the microchamber, the two liquids do notmix, apart from a very negligible mixing, for the purposes of theinvention, being due to a slow process of diffusion, which process inany case begins only once the microchamber CH1 is filled and when acondition of stationary state has been reached in the two liquids.

As illustrated in FIG. 1, an apparatus is used including a singlemicrochamber CH1 having an inlet orifice or input I1 and an outletorifice or output O1 located at the opposite ends of the microchamberCH1, the floor or base of which, parallel to the plane of the sheet inFIG. 1, is composed of a substratum (chip) provided with an array ofelectrodes, eventually provided with sensors.

When the electrodes are energised, and the force (F) is activated whichacts only on the particles (BEADS) present in suspension in the firstliquid but leaves substantially immobile both the first and the secondliquid, the particles (BEADS) are transferred (FIG. 1 d) from the firstliquid (L1) to the second (L2).

For example the first liquid could be a culture medium, and the second aphysiological solution.

The operation may be repeated again, transferring the particles intofurther liquids (L3, . . . LN) in a way corresponding to multiplewashing as usually carried out with test tubes and centrifuges.

However, with centrifuges the starting buffer is diluted in thedestination buffer, for which reason it is often necessary to performtwo or three washes so as to obtain a relative absence of the originalliquid.

In the present invention, the contamination of the destination buffer(L2, . . . Ln) may be greatly limited if the movement of the particles(BEADS) is relatively rapid with respect to the times of diffusion ofthe molecules between the initial and the destination buffer. Thesediffusion times may be made relatively long with suitable stratagems,for example the one illustrated in FIG. 2, which shows the use, for theexecution of the method of the invention, of an apparatus including twomicrochambers CH1, CH2, each provided with a floor having an array ofelectrodes that may be selectively energised and that are hydraulicallyconnected to each other by a restriction or orifice G1,2 and each ofwhich includes an inlet orifice or feed I1, I2, and an outlet orifice ordischarge O1, O2, located at opposite ends of each microchamber CH1, CH2and in a direction perpendicular to the one passing through the orificeG1,2.

While in the case of the apparatus in FIG. 2 the liquids L1 and L2 maybe introduced in laminar movement in predetermined regions of themicrochambers CH1, CH2 until the microchamber CH1 is totally filled withthe liquid L1 and the microchamber CH2 with the liquid L2, thenactivating the electrodes so as to transfer the particles from theliquid L1 (and from the respective microchamber CH1) into the liquid L2and the respective microchamber CH2, guiding them along the orificeG1,2, where there is the interface between the liquids L1, L2 which issmall and therefore limits diffusive phenomena to a minimum, in the caseof the apparatus in FIG. 1 it is first necessary to introduce the liquidL2 into the microchamber CH1, operating in laminar movement, through theorifice I1, without totally filling the microchamber CH1, but only afirst region of the same (for example about half the volume of themicrochamber CH1) situated close to I1 and, later, introduce through thesame orifice I1 the liquid L1 with the suspended particles, stilloperating in laminar movement, so that the liquid L1 pushes the liquidL2 to occupy a second region of the microchamber CH1 situated near theoutlet orifice O1, while the liquid L1 (with the suspended particles)occupies the first region of the microchamber CH1.

In any case, the particles suspended in the washing liquid L2 may berecovered by extracting the liquid L2 from the microchamber or region ofthe microchamber that it occupies, still working in laminar movement,through the orifice O1 (FIG. 1) or O2 (FIG. 2).

The method described substantially contemplates the phases of:

Introducing, in laminar flow conditions, first particles suspended in afirst fluid (for example composed of a liquid, or a semi-liquid), into afirst region of at least one microchamber.

Introducing, in laminar flow conditions, at least one second fluid (forexample a liquid or a semi-liquid) into at least one second region ofsaid at least one microchamber, so as not to mix said at least onesecond fluid with said at least one first fluid.

Activating in said at least one microchamber at least one field of force(F) acting on said particles, to provoke a shift of the particles alonetowards said at least one second region of said at least onemicrochamber containing said at least one second fluid so as to suspendsaid particles in said second fluid.

Optionally (if no other process steps are necessary in the integrateddevice):

Selectively recovering said second fluid and said particles byextracting from said at least one microchamber said second fluidcontaining said suspended particles.

Method for the Incubation of Particles

Reference is made to FIG. 3.

In some ways the method is similar to the previous one, but it alsocontemplates the fact of returning the particles towards the startingbuffer or towards a third destination buffer.

This is a typical problem linked with the process steps necessary forexample to mark cells with antibodies (for example for membranereceptors) eventually coupled with microbeads (for example polystyreneparticles) so as to immunodecorate the cells, with fluorescent tracingagents (for example intracellular dyes such as Calcein, FITC, acridineorange, Trypan Blue), or to fix cells (e.g. with paraformaldehyde), orto lysate certain cells selectively (e.g. exposing the cells to aceticacid, which lysates the red blood cells but not the leucocytes).

In these cases it is important not only to transfer the particles into asecond liquid, but also to keep them in this second liquid for adefinite length of time and then transfer them into a third liquid,which could also be the first (therefore returning the particles to thefirst liquid).

According to the present invention it is therefore possible to transferthe cells into a second liquid where they are exposed to suitablereagents, and then transfer them to a further liquid without reagents.

It is to be noted that, contrary to what can be done with test tubes andcentrifuges, using the method of the invention it is possible to exposethe particles to the reagent also for very short times.

In practice, the BEAD particles suspended in the first liquid (orgeneral fluid) L1 (FIG. 3 b) are introduced in laminar movement into afirst microchamber CH1 of a manipulation apparatus of the type with twomicrochambers having the floor composed of arrays of addressableelectrodes, hydraulically connected to each other by an orifice G1,2,identical to the apparatus illustrated in FIG. 2. Next (orsimultaneously), operating in conditions of laminar movement, thegeneral fluid or liquid L2 is introduced into the second microchamberCH2 (FIG. 3 c) and the electrodes are then selectively activated tocreate a field acting with force F on the particles in such a way as totransfer them, through the orifice G1,2, from the microchamber CH1 tothe microchamber CH2 without producing the shift of the liquids L1,L2(FIG. 3 d), which at this point are in stationary conditions; and then,after a predetermined incubation time (which may also be short aspreferred), the particles are transferred by the force F back into themicrochamber CH1 (FIG. 3 e) which may still contain the starting liquidL1, or a new liquid (fluid) L3 . . . Ln which in the meantime has beenintroduced into the microchamber CH1 through I1 after draining theliquid L1, for example through O1. Lastly the particles may be takentogether with the liquid present in CH1 and in which they are suspended(FIG. 3 f) through either O1 or I1, always operating in laminarmovement.

The method described substantially contemplates the phases of:

Introducing, in laminar flow conditions, said particles suspended in afirst fluid into a first region of at least one microchamber of anapparatus for manipulating particles;

Introducing, in laminar flow conditions, at least one second fluid intoat least one second region of said at least one microchamber, so as notto mix said at least one second fluid with said at least one firstfluid.

Activating in said at least one microchamber at least one field of force(F) acting on said particles, to provoke a shift of the particles alonein a predetermined direction into said at least one second region ofsaid at least one microchamber containing said at least one secondfluid, so as to suspend said particles in said second fluid.

Activating in said at least one microchamber at least one field of force(F) acting on said particles, to provoke a shift in the oppositedirection to the previous one of the particles alone so as to bring themback into said at least one first region of said at least onemicrochamber.

Optionally, the method of the invention may also comprise the phases of:

Replacing said first fluid in said at least one first region of said atleast one microchamber with at least one third fluid, before bringingsaid particles back into said at least one first region of said at leastone microchamber;

Selectively recovering said particles by extracting from said at leastone microchamber the fluid in which the particles are suspended.

Method for Carrying Out Complex Protocols

The washing and incubation operations may be composed in sequence,realising complex protocols. For example with the following phases:

1. Shifting the cells from the liquid Li to a different destinationliquid Li+12. Waiting a period of time Ti+13. Shifting the cells from the liquid Li+1 to a further liquid Li+24. Repeating the preceding phases 2) and 3) until the cells or particlesare suspended in a liquid Li+n.

For example the cells could be:

-   -   introduced suspended in the culture medium,    -   shifted into a region of the space occupied by a washing liquid        (such as physiological solution),    -   transferred into a region of the space containing        paraformaldehyde,    -   kept in this reagent for the time necessary to obtain the fixing        and permeabilisation of the membrane,    -   washed by transferring them to a further region of the space        containing physiological solution,    -   transferred into a region of the space containing proteins with        fluorescent markers in order to recognise proteins potentially        present inside the cell,    -   washed by transferring them to a further region of the space        containing physiological solution,    -   analysed with sensors to recognise which cells express the        proteins identified by the fluorescent marker.

According to this aspect of the invention, a method is thereforeperformed for carrying out complex experimental protocols on particleskept suspended in a fluid, comprising the phases of:

i) Suspending said particles in a first process fluid (Li) confined in afirst region of space;ii) Selectively shifting said particles into a second process fluid(Li+1) confined in a second region of space, maintaining the first fluidand the second fluid in stationary conditions, by applying to saidparticles a force F obtained by activating a field of force actingexclusively on said particles;iii) Repeating phase ii) a number n of times until said particles aresuspended in a final fluid (Li+n), so as to contact with said particlesa plurality of different process fluids, at least some of which are ableto interact with said particles;iv) Analysing said particles with sensors located in a region of spacecontaining said final fluid in a confined way.Method for Studying the Dynamics of Reaction of Particles with Reagents

Contrary to what is possible also with techniques based on flowMicrosystems such as the already mentioned Seger et al. LabChip, thetime that the particles remain in the incubating reagent is not linkedto the flow dynamics of the sample.

Moreover, every single particle may be exposed to the reagent fordifferent times, even though it is manipulated in the same device. Infact it is sufficient to locate said particle in the region of spaceoccupied by the reagent for a certain time Ti, and then bring it backinto the final destination suspension buffer.

It is thus possible to expose N particles to a reagent for T1-TNdifferent times.

It is therefore possible to study characteristics dynamically (withrelation to time), such as for example the uptake of the reagent or thevariation of characteristics that it causes on the cell.

This may occur with external optical sensors or preferably, usingsensors integrated in the chip defining the floor of the microchambersCH1, . . . CHn used to keep confined the n process liquids with which tocontact selectively the particles studied. The integrated sensor may bepreferentially optical and/or impedenziometric. One may for example:

-   -   Measure the uptake of a dye (for example trypan blue)    -   Measure the uptake of a fluorochrome (for example FITC, acridine        orange etc.)    -   Measure the uptake of Calcein by increasing the fluorescence of        a cell (Calcein is fluorescent only inside the cell);    -   Measure the variation of volume of a cell;    -   Check the lysis of a cell exposed to a reagent.

All this information can in fact be found in an integrated way accordingto the method of the prior art or, better, according to the methodsdescribed in the international patent application in the name of thesame Applicant, previously mentioned.

This method, which is a variation of the one previously explained,therefore contemplates that the phases ii) and iii) described above arecarried out in such a way as to expose a number N of particles to atleast one predetermined reagent for T1, . . . TN different times; andthat the phase iv) contemplates the dynamic study, with relation totime, of variable characteristics of said particles, such as for examplethe uptake of reagent or the variation of properties of the particleslinked to reagent uptake.

Method for Studying the Secretion of Particles

Besides the characteristics of the cell, in terms of reagent uptake orthe variation of physical properties, it is possible to study thesecretion of single cells. To do this, the cells are moved towards areaction buffer which contains substances that are modified depending onthe presence of the type of molecules of which the secretion is to bestudied.

If the secretion dynamics is sufficiently rapid with respect to thecoefficient of diffusion of the secretion in the liquid, by analysingthe liquid in the vicinity of the cell to be analysed with suitablesensors (for example optical sensors outside the chip defining the floorof the microchambers in which the reactions take place, or integrated inthe chip, or impedenziometric sensors integrated in the chip), the cellsthat produce more or less secretion can be identified. In the prior art,numerous methods are available for revealing secreted substances. Thesemethods may be used with few or no modifications in order to study thesecretion, not of a group but, according to the invention, of singlecells.

As an example without limitation, one may mention the use ofchemiluminescent substances, of fluorescent substances with quenchingwhich is inhibited by the presence of a certain secretion, or substanceswhich change colour in the presence of a certain secretion.

If the diffusion of the secretion is too fast with respect to thedynamics of secretion and to the time scale for which the cells are tobe analysed, it is necessary to adopt more elaborate solutions.Otherwise, the signal represented by the variation of colour,fluorescence, impedance or light intensity emitted, is not confined toan area around the cell and does not allow the separation of the signalcoming from different cells.

In this case, the method comprises the phase of encapsulating the cellsin porous/gelatinous substances which keep the secretion longer, slowingdown its coefficient of diffusion and preventing the overlapping of thesignal from different cells.

This method may be used for example in the selection of encapsulatedcells to avoid rejection in transplants for therapeutic purposes.

The method just described therefore contemplates that the phases ii) andiii) described above are performed in such a way as to expose apredetermined number of particles, individually or in a group, to atleast one predetermined reagent that is able to interact with anysubstances secreted by said particles; while phase iv) contemplates atthis point the identification of this interaction with sensors,integrated or not. Moreover, it may optionally contemplate the phase ofencapsulating at least some particles in porous/gelatinous substancesable to keep back any substances secreted by the particles or at leastto slow down their diffusion in the fluid in which the particles aresuspended so as to prevent the overlapping of signals generated by thesensors in reply to the presence of different particles.

Method for Studying the Effects of the Exposure of Particles to Reagents

As well as the reaction dynamics, also the dynamics of the reactions ofthe exposure of a cell to a determined reagent may be analysed with hightime resolution and above all with low delay.

By returning the single particles to a neutral buffer, the modificationsgenerated by the exposure of the cells to the reagent may be studiedafter a variable period of time.

As above, this may be done with external optical sensors, or preferablewith integrated optical or impedenziometric sensors.

For example one might want to check the expression of a reporter gene,or cellular differentiation.

Method for Selecting Particles

As well as observing the dynamics of reaction with a reagent, andobserving the effects of the exposure to said reagent, it is possible tomake an automatic selection of the cells based on the response of thesame to the stimuli applied (in the form of reagents).

For example this allows the selection of the cells which, from a certainpoint of view, are more or less affected by a determined stimulus, orseries of stimuli.

For example one might want to isolate the cells which absorb a smallerquantity of drugs of one type, or the cells that produce a greaterquantity of a certain protein once exposed to a reagent.

So, according to this aspect of the invention, the phase is contemplatedof selecting, in response to a signal detected by the sensors, theparticles to which the generation of the signal is related; thisselection phase is then carried out applying only to the particlesrelated to the generation of that signal the force F generated by thefield of forces that may be generated by means of the selectiveactivation of the array of electrodes defining the floor of themicrochambers to transport said particles and only them into a definedconfined region of space, for example composed of a predeterminedmicrochambers CHn or of a predetermined region of volume of the samemicrochamber in which the sensors are situated.

Method for “Constructing” Particles

By means of the methods described so far it is clear that one can alsoshift a particle or a group of “seed” particles from a first liquid toat least one second liquid, in which said particle(s) is (are) modifiedfor example in one of the following ways: coating itself (for examplewith substances present in the second liquid or which are the product ofthe reaction between the particle and a reagent contained in the secondliquid, growing (typically in the case of biological particles, such ascells, which may for example draw nourishment or a multiplication signalfrom the second liquid), soaking (typically in the case of artificialporous particles, such as microspheres).

The particle is then moved into a possible third, fourth, n-th reagent,realising a complex protocol to supply at the end “product” particles,for example composed of layers with controlled dimensions of differentmaterials.

According to this aspect of the invention, a method is thereforeprovided for realising particles having a complex predeterminedstructure starting from first particles with a simple structure,comprising the phases of:

i) Suspending said first particles in a first process fluid (Li)confined in a first region of space;ii) Selectively shifting said particles with a simple structure into asecond process fluid (Li+1) confined in a second region of space,maintaining the first fluid and the second fluid in stationaryconditions, by applying to said particles a force F obtained byactivating a field of force acting exclusively on said particles;iii) Repeating phase ii) a number n of times until said first particlesare suspended in a final fluid (Li+n), so as to contact with said firstparticles a plurality of different process fluids, at least some ofwhich are able to interact with said first particles in one of the wayschosen in the group comprising: coating, growing, soaking.

Apparatus for the Complex Processing of Particles

In the case of multi-step protocols, which require a sequence of cellprocessing steps, it is particularly useful to realise a manipulatingapparatus as schematically illustrated in FIG. 4, based on threemicrochambers CH1,CH2,CH3 arranged in sequence and realised as describedpreviously for the microchambers CH1,CH2 in FIGS. 1-3, in which thecentral microchamber CH2 is hydraulically connected to the other twothrough two orifices G1,2 and G2,3 of hydraulic communication in thedirection of the sequence of microchambers, which are located not onlyon opposite sides (in the direction of the sequence of microchambers) ofthe microchamber CH2, but are also offset to each other in thetransverse sense, that is in a direction perpendicular to the directionof arrangement in sequence of the microchambers, being located one closeto the inlet orifice I2 of the microchamber CH2 and the other close tothe outlet orifice O2 of the microchamber CH2.

The central microchamber (CH2) is used for washing the cells between thebuffer contained in the starting microchamber (CH1) and the reagentmicrochamber (CH3).

The first advantage of this device is that the distance between theorifices of hydraulic communication that constitute the two doors (G1,2)between the first and the second microchamber and (G2,3) between thesecond and the third microchamber increases the time necessary for thediffusion to contaminate the first starting liquid with the reagent inthe third microchamber (CH3). Moreover, if after the passage of thecells washing buffer is flushed constantly into the second microchamber(CH2), any contamination between the reagent present in the thirdmicrochamber and the initial buffer is avoided.

Preferably, during flushing, the inlets and outlets (11,13 and O1,O3) ofthe other microchambers are kept closed, so as to keep the flow confinedinside the second microchamber.

The liquids are preferably introduced in this order:

CH1, CH3, CH2. In this way, as long as the meniscuses of the liquids L1and L3 (that is the ones contained respectively in the microchambers CH1and CH3) look onto the microchamber CH2, but do not touch, there is nocontamination between the two due to diffusion.

Arbitrarily long multi-step reactions may be also be completed with thisapparatus by completely replacing the buffer in the first microchamber(CH1) while the cells are in the third microchamber (CH3) andvice-versa. In this way the reagents are “multiplexed” in the space.

Alternatively a device may be realised in which all the reagents areinjected in the initial phase and are already present at the start ofthe manipulation of the particles. This apparatus is shown in FIG. 5.

It comprises a first and a second terminal microchamber (CH1,CHn) and aplurality of intermediate microchambers (CH2, . . . CHn-1) arranged insequence between the terminal microchambers and each of which ishydraulically connected to the microchamber immediately before andimmediately after in the direction of the sequence of microchambers bymeans of a respective first and second orifice (G1,2; Gn-1,n) ofhydraulic communication in the direction of the sequence ofmicrochambers, offset to each other in a transverse direction to thedirection of the sequence of microchambers.

Generalising what has been said previously, the apparatus in FIG. 5 ispreferably filled firstly by filling the microchambers with an evenindex and secondly the microchambers with an odd index, so as to avoidcontacts and contaminations until the liquids come into contact.

1.-8. (canceled)
 9. Method for carrying out complex experimentalprotocols on particles kept suspended in a fluid, comprising phases: i)Suspending said particles in a first process fluid (Li) confined in afirst region of space; ii) Selectively shifting said particles into asecond process fluid (Li+1) confined in a second region of space,maintaining the first fluid and the second fluid in stationaryconditions, by applying to said particles a force F obtained byactivating a field of force acting exclusively on said particles; iii)Repeating phase ii) a number n of times until said particles aresuspended in a final fluid (Li+n), so as to contact with said particlesa plurality of different process fluids, at least some of which are ableto interact with said particles;
 10. Method according to claim 9,further comprising a phase: iv) analyzing said particles with sensorslocated in a region of space containing said final fluid in a confinedway.
 11. Method according to claim 10, wherein phases ii) and iii) arecarried out in such a way as to expose a number N of particles to atleast one predetermined reagent for T1, . . . TN different times; saidphase iv) contemplating the dynamic study, with relation to time, ofvariable characteristics of said particles, such as for example theuptake of reagent or the variation of properties of the particles linkedto reagent uptake.
 12. Method according to claim 10, wherein phases ii)and iii) are performed in such a way as to expose a predetermined numberof particles, individually or in a group, to at least one predeterminedreagent that is able to interact with any substances secreted by saidparticles; said phase iv) contemplating the identification of saidinteraction with said sensors.
 13. Method according to claim 12, whereinthe phase of encapsulating at least some said particles inporous/gelatinous substances able to keep back any substances secretedby said particles or at least to slow down their diffusion in the fluidin which said particles are suspended so as to prevent the overlappingof signals generated by said sensors in reply to the presence ofdifferent particles.
 14. Method according to claim 10, comprising thephase of selecting, in response to a signal detected by said sensors,the particles to which the generation of said signal is related; saidselection phase being carried out applying only to the particles relatedto the generation of said signal said force F generated by said field offorces to transport said particles into a defined confined region ofspace.
 15. Method for realising particles having a complex predeterminedstructure starting from first particles with a simple structure,comprising phases: i) Suspending said first particles in a first processfluid (Li) confined in a first region of space; ii) Selectively shiftingsaid particles with a simple structure into a second process fluid(Li+1) confined in a second region of space, maintaining the first fluidand the second fluid in stationary conditions, by applying to saidparticles a force F obtained by activating a field of force actingexclusively on said particles; iii) Repeating phase ii) a number n oftimes until said first particles are suspended in a final fluid (Li+n),so as to contact with said first particles a plurality of differentprocess fluids, at least some of which are able to interact with saidfirst particles in one of the ways chosen in the group comprising:coating, growing, soaking.
 16. Apparatus for carrying out complexprocessing of particles, of the type comprising microchambers, each oneof which is delimited by a plane or floor composed of a chip,hydraulically connected to each other by at least one restriction ororifice and each of which includes an inlet orifice (11,12) and anoutlet orifice (01,02) located at opposite ends of each microchamber ina direction perpendicular to that of the hydraulic connection betweenthe microchambers; characterised in that it comprises a first and asecond terminal microchamber (CH1, CH3) and at least one intermediatemicrochamber (CH2) arranged in sequence with each other, wherein said atleast one intermediate microchamber (CH2) is connected to said terminalmicrochambers through a first and a second orifice (G1, 2; G2,3) ofhydraulic communication in the direction of the sequence ofmicrochambers, and offset with each other in a transverse direction, ina direction perpendicular to the direction of arrangement in sequence ofthe microchambers, being located one in the vicinity of the inletorifice (12) and the other in the vicinity of the outlet orifice (02) ofsaid at least one intermediate microchamber (CH2).
 17. Apparatusaccording to claim 16, comprising a plurality (CH2, . . . CHn-I) ofintermediate microchambers arranged in sequence between said first andsecond terminal microchamber (CH1, CHn), each said intermediatemicrochamber being hydraulically connected to the microchamberimmediately before and immediately after in the direction of thesequence of microchambers by means of a respective first and secondorifice (G1, 2; Gn-I, n) of hydraulic communication in the direction ofthe sequence of microchambers, offset to each other in a transversedirection to the direction of the sequence of microchambers. 18.Apparatus according to claim 16, wherein said plane or floor of saidchip comprises an array of electrodes that may be selectively energized,able to generate said force (F).
 19. Apparatus according to claim 16,wherein said plane or floor of said chip comprises an array ofintegrated sensors.